The overall objective of this research proposal is to define the multifaceted role of the MUC4 mucin in the pathogenesis of pancreatic cancer (PC). During past funding cycles, we have made considerable progress, beginning with the cloning of the MUC4 cDNA from human pancreatic tumor cDNA libraries, establishing its complete genomic organization to understanding its function and regulation in PC cells. In our recent studies, we have shown a direct association of MUC4 with the tumorigenic and metastatic pancreatic cancer phenotype and provided experimental evidence for a functional role of MUC4 in growth and metastatic properties of PC cells. We have also demonstrated the oncogenic potential of MUC4 in NIH3T3 mouse fibroblast cells. Interestingly, our studies have also revealed that MUC4 regulates the expression of HER2 by post-transcriptional mechanism(s). We have shown that both MUC4 and HER2 physically interact with each other in PC cells. In this renewal application, we aim to establish the mechanistic basis for the pathological role of MUC4 in PC and to precisely define its cooperative action with other genetic aberrations during the course of pancreatic cancer pathogenesis. We hypothesize that the multi-domain structural features of MUC4 enable it to functionally participate in the early development and malignant process of PC cells via altering the normal cell-cell and cell-ECM (extra-cellular matrix) interactions, while simultaneously forming novel, cancer-specific interactions. To test our hypothesis, we propose three specific aims. In Aim 1, we will investigate the specific domain(s) or motif(s) in MUC4 responsible for its interaction with HER2, and determine the specificity and nature (direct or indirect) of interaction. In addition, we will define the mechanisms by which MUC4 regulates HER2 expression. Aim 2 will investigate the molecular mechanisms implicated in defining the multiple roles of MUC4 in tumor growth and metastasis. In Aim 3, we will investigate the cooperative action of MUC4 in combination with other defined oncogenic mutations during the early development of pancreatic cancer by generating MUC4-transgenic mice with conditional pancreas-specific expression. Additionally, we will investigate the cooperative action of MUC4 and K-rasG12D (oncogenic) in mouse models by cross-breeding. We will also utilize cultured mouse pancreatic ductal cells (PDCs), PDCs harboring K-ras mutations, and/or p53 inactivation to study MUC4 function(s) in syngeneic system. Taken together, these studies will establish the mechanistic basis for the role of MUC4 in early and late stages of pancreatic cancer progression. PUBLIC HEALTH RELEVANCE: The proposed second competitive research program in its 10th year aims to understand the mechanistic basis of the role of MUC4 in the pathogenesis of lethal pancreatic cancer (PC). During previous cycles, studies pertinent to MUC4 structure, function and regulation were performed in PC cells. MUC4 is aberrantly expressed in majority of pancreatic cancer cases compared to no detection in the normal pancreas. The proposed studies will investigate the precise role of MUC4 and its domains using human pancreatic cancer cell lines as well as in transgenic and syngeneic animal models.